The concentrations and distributions of three C-terminal variants of the GLT1 (EAAT2; slc1a2) glutamate transporter protein in rat brain tissue suggest differential regulation

The neurotransmitter glutamate is inactivated by cellular uptake; mostly catalyzed by the glutamate transporter GLT1 (slc1a2, excitatory amino acid transporter [EAAT2]) subtype which is expressed at high levels in brain astrocytes and at lower levels in neurons. Three coulombs-terminal variants of GLT1 exist (GLT1a, GLT1b and GLT1c). Their cellular distributions are currently being debated (that of GLT1b in particular). Here we have made antibodies to the variants and produced pure preparations of the individual variant proteins. The immunoreactivities of each variant per amount of protein were compared to that of total GLT1 immunoisolated from Wistar rat brains. At eight weeks of age GLT1a, GLT1b and GLT1c represented, respectively 90%+/-1%, 6+/-1% and 1%+/-0.5% (mean+/-SEM) of total hippocampal GLT1. The levels of all three variants were low at birth and increased towards adulthood, but GLT1a increased relatively more than the other two. At postnatal day 14 the levels of GLT1b and GLT1c relative to total GLT1 were, respectively, 1.7+/-0.1 and 2.5+/-0.1 times higher than at eight weeks. In tissue sections, antibodies to GLT1a gave stronger labeling than antibodies to GLT1b, but the distributions of GLT1a and GLT1b were similar in that both were predominantly expressed in astroglia, cell bodies as well as their finest ramifications. GLT1b was not detected in nerve terminals in normal brain tissue. The findings illustrate the need for quantitative measurements and support the notion that the importance of the variants may not be due to the transporter molecules themselves, but rather that their expression represents the activities of different regulatory pathways.

[1]  T. Bliss,et al.  ARG3.1/ARC expression in hippocampal dentate gyrus astrocytes: ultrastructural evidence and co-localization with glial fibrillary acidic protein , 2007, Journal of cellular and molecular medicine.

[2]  Masahiko Watanabe,et al.  Indispensability of the glutamate transporters GLAST and GLT1 to brain development , 2006, Proceedings of the National Academy of Sciences.

[3]  V. Haroutunian,et al.  Quantitative analysis of glutamate transporter mRNA expression in prefrontal and primary visual cortex in normal and schizophrenic brain , 2006, Neuroscience.

[4]  K. P. Lehre,et al.  The Number of Glutamate Transporter Subtype Molecules at Glutamatergic Synapses: Chemical and Stereological Quantification in Young Adult Rat Brain , 1998, The Journal of Neuroscience.

[5]  S. Stamm,et al.  Differential regulation of 5′ splice variants of the glutamate transporter EAAT2 in an in vivo model of chemical hypoxia induced by 3‐nitropropionic acid , 2003, Journal of neuroscience research.

[6]  Lin Jin,et al.  Aberrant RNA Processing in a Neurodegenerative Disease: the Cause for Absent EAAT2, a Glutamate Transporter, in Amyotrophic Lateral Sclerosis , 1998, Neuron.

[7]  A. Ludolph,et al.  Splicing of the glutamate transporter EAAT2: a candidate gene of amyotrophic lateral sclerosis , 1998, Journal of neurology, neurosurgery, and psychiatry.

[8]  J. Storm-Mathisen,et al.  Differential distribution of the glutamate transporters GLT1 and rEAAC1 in rat cerebral cortex and thalamus: an in situ hybridization analysis , 1997, Anatomy and Embryology.

[9]  M. Brasch,et al.  Interaction between the glutamate transporter GLT1b and the synaptic PDZ domain protein PICK1 , 2007, The European journal of neuroscience.

[10]  J. Storm-Mathisen,et al.  Glutamate transporters in glial plasma membranes: Highly differentiated localizations revealed by quantitative ultrastructural immunocytochemistry , 1995, Neuron.

[11]  D. Troost,et al.  Impaired RNA splicing of 5′-regulatory sequences of the astroglial glutamate transporter EAAT2 in human astrocytoma , 2001, Journal of neurology, neurosurgery, and psychiatry.

[12]  K. P. Lehre,et al.  A monoclonal antibody raised against an [Na+K+]coupled l‐glutamate transporter purified from rat brain confirms glial cell localization , 1993, FEBS letters.

[13]  E. Gouaux,et al.  Structure of a glutamate transporter homologue from Pyrococcus horikoshii , 2004, Nature.

[14]  M. Hediger,et al.  Primary structure and functional characterization of a high-affinity glutamate transporter , 1992, Nature.

[15]  U. Berger,et al.  Distribution of the glutamate transporters GLAST and GLT‐1 in rat circumventricular organs, meninges, and dorsal root ganglia , 2000, The Journal of comparative neurology.

[16]  J. Storm-Mathisen,et al.  Glutamate : transmitter in the central nervous system , 1981 .

[17]  S. Stamm,et al.  Alternative splicing of the 5′‐sequences of the mouse EAAT2 glutamate transporter and expression in a transgenic model for amyotrophic lateral sclerosis , 2002, Journal of neurochemistry.

[18]  R. Sullivan,et al.  A new GLT1 splice variant: cloning and immunolocalization of GLT1c in the mammalian retina and brain , 2004, Neurochemistry International.

[19]  P. Kugler,et al.  Complementary neuronal and glial expression of two high-affinity glutamate transporter GLT1/EAAT2 forms in rat cerebral cortex , 2003, Histochemistry and Cell Biology.

[20]  Y. Kanai,et al.  Tissue specific variants of glutamate transporter GLT‐1 , 1997, FEBS letters.

[21]  K. P. Lehre,et al.  Localization of transporters using transporter-specific antibodies. , 1998, Methods in enzymology.

[22]  U. Berger,et al.  Comparative analysis of glutamate transporter expression in rat brain using differential double in situ hybridization , 1998, Anatomy and Embryology.

[23]  R. Sullivan,et al.  Distribution of two splice variants of the glutamate transporter GLT‐1 in rat brain and pituitary , 2002, Glia.

[24]  A. Ludolph,et al.  Alternative splicing of the glutamate transporter EAAT2 (GLT-1) , 1998, Neuroscience Letters.

[25]  K. P. Lehre,et al.  Brain Glutamate Transporter Proteins Form Homomultimers* , 1996, The Journal of Biological Chemistry.

[26]  Olaf Riess,et al.  Expression mapping of tetracycline-responsive prion protein promoter: Digital atlasing for generating cell-specific disease models , 2006, NeuroImage.

[27]  P. Rosenberg,et al.  Cellular and subcellular mRNA localization of glutamate transporter isoforms GLT1a and GLT1b in rat brain by in situ hybridization , 2005, The Journal of comparative neurology.

[28]  Trygve B. Leergaard,et al.  Toward a workbench for rodent brain image data systems architecture and design , 2007, Neuroinformatics.

[29]  J. Storm-Mathisen,et al.  Differential expression of two glial glutamate transporters in the rat brain: quantitative and immunocytochemical observations , 1995, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[30]  K. P. Lehre,et al.  Specificity controls for immunocytochemistry , 2006, Anatomy and Embryology.

[31]  C. Aoki,et al.  The Glutamate Transporter GLT1a Is Expressed in Excitatory Axon Terminals of Mature Hippocampal Neurons , 2004, The Journal of Neuroscience.

[32]  Rachel Blitzblau,et al.  Expression of a Variant Form of the Glutamate Transporter GLT1 in Neuronal Cultures and in Neurons and Astrocytes in the Rat Brain , 2002, The Journal of Neuroscience.

[33]  R. D. O'Shea,et al.  Transporters for L‐glutamate: An update on their molecular pharmacology and pathological involvement , 2007, British journal of pharmacology.

[34]  K. Lesch,et al.  A splice variant of glutamate transporter GLT1/EAAT2 expressed in neurons: cloning and localization in rat nervous system , 2002, Neuroscience.

[35]  B. Cubelos,et al.  The glutamate transporter GLT1b interacts with the scaffold protein PSD‐95 , 2008, Journal of neurochemistry.

[36]  E. Asan,et al.  Expression of the glutamate transporter GLT1 in neural cells of the rat central nervous system: Non-radioactive in situ hybridization and comparative immunocytochemistry , 1996, Neuroscience.

[37]  T. Storck,et al.  Structure, expression, and functional analysis of a Na(+)-dependent glutamate/aspartate transporter from rat brain. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[38]  D. Attwell,et al.  A quantitative assessment of glutamate uptake into hippocampal synaptic terminals and astrocytes: New insights into a neuronal role for excitatory amino acid transporter 2 (EAAT2) , 2008, Neuroscience.

[39]  Georg Grön,et al.  The RNA of the glutamate transporter EAAT2 is variably spliced in amyotrophic lateral sclerosis and normal individuals , 1999, Journal of the Neurological Sciences.

[40]  D. Bergles,et al.  Specificity of antibodies: Unexpected cross-reactivity of antibodies directed against the excitatory amino acid transporter 3 (EAAT3) , 2005, Neuroscience.

[41]  J. Rozyczka,et al.  Multiple 5'-splice variants of the rat glutamate transporter-1. , 2005, Brain research. Molecular brain research.

[42]  J. Storm-Mathisen,et al.  An [Na+ + K+]coupledl-glutamate transporter purified from rat brain is located in glial cell processes , 1992, Neuroscience.

[43]  A. Levey,et al.  Localization of neuronal and glial glutamate transporters , 1994, Neuron.

[44]  Lingzhi Fan,et al.  The glucose oxidase-DAB-nickel method in peroxidase histochemistry of the nervous system , 1988, Neuroscience Letters.

[45]  M. Bellesi,et al.  Synaptic localization of GLT‐1a in the rat somatic sensory cortex , 2009, Glia.

[46]  E. Seeberg,et al.  Differential Expression of Two Glial Glutamate Transporters in the Rat Brain: an In Situ Hybridization Study , 1994, The European journal of neuroscience.

[47]  E. Gouaux,et al.  Trimeric subunit stoichiometry of the glutamate transporters from Bacillus caldotenax and Bacillus stearothermophilus. , 2003, Biochemistry.

[48]  C. Shaw,et al.  Late appearance of glutamate transporter defects in a murine model of ALS–parkinsonism dementia complex , 2007, Neurochemistry International.

[49]  B. McEwen,et al.  Chronic restraint stress up-regulates GLT-1 mRNA and protein expression in the rat hippocampus: Reversal by tianeptine , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[50]  L. Honig,et al.  Glutamate transporter EAAT2 splice variants occur not only in ALS, but also in AD and controls , 2000, Neurology.

[51]  Masahiko Watanabe,et al.  Epilepsy and exacerbation of brain injury in mice lacking the glutamate transporter GLT-1. , 1997, Science.

[52]  F. Kirchhoff,et al.  Segregated Expression of AMPA-Type Glutamate Receptors and Glutamate Transporters Defines Distinct Astrocyte Populations in the Mouse Hippocampus , 2003, The Journal of Neuroscience.

[53]  A. Ludolph,et al.  5′‐Heterogeneity of the human excitatory amino acid transporter cDNA EAAT2 (GLT‐1) , 1998, Neuroreport.

[54]  Y. Itoyama,et al.  Identification of alternative splicing forms of GLT-1 mRNA in the spinal cord of amyotrophic lateral sclerosis patients , 1998, Neuroscience Letters.

[55]  D. Graham,et al.  Interindividual differences in the levels of the glutamate transporters GLAST and GLT, but no clear correlation with Alzheimer's disease , 1999, Journal of neuroscience research.

[56]  Matthew E. R. Butchbach,et al.  Human Glioma Cells and Undifferentiated Primary Astrocytes That Express Aberrant EAAT2 mRNA Inhibit Normal EAAT2 Protein Expression and Prevent Cell Death , 2002, Molecular and Cellular Neuroscience.

[57]  R. Sullivan,et al.  Cloning, transport properties, and differential localization of two splice variants of GLT‐1 in the rat CNS: Implications for CNS glutamate homeostasis , 2004, Glia.

[58]  J. Storm-Mathisen,et al.  Differential Developmental Expression of the Two Rat Brain Glutamate Transporter Proteins GLAST and GLT , 1997, The European journal of neuroscience.

[59]  J. G. Bjaalie,et al.  Database and tools for analysis of topographic organization and map transformations in major projection systems of the brain , 2005, Neuroscience.

[60]  E. Seeberg,et al.  Cloning and expression of a rat brain L-glutamate transporter , 1992, Nature.

[61]  J. Rothstein,et al.  Glutamate Transporter Protein Subtypes Are Expressed Differentially during Rat CNS Development , 1997, The Journal of Neuroscience.

[62]  G. Jansen,et al.  Alternative splicing of glutamate transporter EAAT2 RNA in neocortex and hippocampus of temporal lobe epilepsy patients , 2004, Epilepsy Research.

[63]  S. Stamm,et al.  Differential RNA cleavage and polyadenylation of the glutamate transporter EAAT2 in the human brain. , 2000, Brain research. Molecular brain research.

[64]  T. Rauen,et al.  Electrogenic Glutamate Transporters in the CNS: Molecular Mechanism, Pre-steady-state Kinetics, and their Impact on Synaptic Signaling , 2004, The Journal of Membrane Biology.

[65]  Clifford B Saper,et al.  Magic peptides, magic antibodies: Guidelines for appropriate controls for immunohistochemistry , 2003, The Journal of comparative neurology.